(a) Field of the Invention
The invention relates to Replication Protein A (RPA) and more particularly to a RPA transcriptional factor to treat neoplastic disorders such as cancer.
(b) Description of Prior Art
Replication Protein A (RPA), also known as replication factor A (RFA), is a ubiquitous and abundant heterotrimeric protein required for DNA replication, repair and recombination in eukaryotes. RPA nonspecifically binds single-stranded DNA and plays an essential role in the regulation of DNA metabolism via multiple protein interactions and/or RPA phosphorylation. More particularly, RPA binds single-stranded DNA with strong affinity (association constant of 109–1011 M−1) and greatest affinity for polypyrimidine tracts. RPA also binds double-stranded DNA with lower affinity and is likely to facilitate DNA unwinding. RPA may play a role in the regulation of transcription by binding regulatory elements in promoters; in yeast, RPA binds specific regulatory sequences in the promoters of DNA repair and metabolism genes (Singh K. et al., 1995, Proceedings of the National Academy of Science USA 92(11):4907–11).
RPA is made of three subunits: a 70-kDa subunit (RPA70), a 32-kDa middle subunit (RPA32) and a 14-kDa subunit (RPA14). The RPA32 subunit is phosphorylated in a cell cycle-dependent manner.
RPA-protein interactions appear to be largely mediated by the large 70-kDa subunit (RPA70). RPA70 interacts with the p53, GAL4, VP16, EBNA1 and SV40T antigens and with DNA polymerase alpha (Wold, M., 1997, Annual Review of Biochemistry, “Replication Protein A: A Heterotrimeric, Single-Stranded DNA-binding Protein Required for Eukaryotic DNA Metabolism”). It is also important in interaction with DNA repair proteins involved in damage recognition and excision.
Interaction with XPF stimulates its 5′ junction-specific endonuclease activity, interaction with XPG targets this endonuclease to damaged DNA, and interaction with ERCC1 (ERCC1 also binds xeroderma pigmentosum group A factor, XPA, which is another NER factor) promotes exonuclease activity.
The possibility of interaction by the aforementioned repair proteins with RPA32 has not been clearly elucidated. However, interactions with some proteins involved in DNA repair appear to be mediated by RPA32, such as interaction with XPA and uracil-DNA glycosylase. A region of significant homology between uracil-DNA glycosylase and XPA was also reported, suggestive of the possibility of a common binding motif to RPA32 across several different proteins. Furthermore, some important protein interactions, such as with RAD52, appear to involve all three subunits of RPA (Hays, S. et al., 1998, Molecular and Cellular Biology 18(7):4400–4406).
In cells, RPA is phosphorylated by DNA-dependent protein kinase (DNA-PK) when RPA is bound to single-strand DNA, during the S phase and after DNA damage; and also possibly by ATM.
Phosphorylation of RPA is observed in a cell-cycle dependent manner and in response to DNA damage (i.e. UV light, X-rays, cisplatin) in eukaryotic systems. This phosphorylation takes place predominantly on the N-terminal region of RPA32 and was previously thought to be effected by DNA-dependent protein kinase (DNA-PK). However, RPA hyperphosphorylation still takes place in SCID cells where DNA-PK is believed to be responsible for its repair and recombination defects. Ataxia telangiectasia mutated gene (ATM), an important cell cycle checkpoint protein kinase belonging to the same kinase family as DNA-PK, may be responsible for the in vivo phosphorylation of RPA32. In Saccharomyces cerevisiae, the ATM homolog, MEC1, is essential for RPA phosphorylation. Furthermore, ionizing radiation-induced phosphorylation of RPA32 is deficient and reduced in primary fibroblasts from patients suffering from ataxia telangiectasia in comparison to normal, aged fibroblasts.
The result of RPA32 phosphorylation on DNA metabolism is largely unsolved. It has been noted that IR-induced RPA phosphorylation can be uncoupled from the S-phase checkpoint in ataxia telangiectasia cells, suggesting that RPA phosphorylation in itself is not necessary or sufficient for an S-phase arrest. Phosphorylation, however, may affect the conformation of RPA, thereby modulating its affinity for DNA and its protein interactors, and altering the balance between DNA replication and repair. Hyperphosphorylation of RPA32 in vivo is concordant with a decrease in the binding of RPA to single-stranded DNA. This observation is interesting to note since phosphorylated RPA32 is found predominantly in the S-phase of the cell cycle.
RPA has been found to have a high affinity for UV-damaged and cisplatin-damaged DNA and the accompanying phosphorylated form of RPA is correlated strongly with a reduction of the in vitro DNA replication activity of the concerned cell extracts.
It would therefore be highly desirable to identify physiologically relevant protein interactors of the RPA32 subunit of Replication Protein A. Identification of such protein interactors would contribute to the understanding of DNA repair, transcription, and cell signaling. The proteins involved in nucleotide excision repair (NER), for example, are quite numerous and the basis for their interaction and function is not yet completely understood. Understanding the regulation of these pathways would assuredly lend insight into their role in cancer susceptibility. RPA, as a protein involved integrally in modulating DNA repair, replication and recombination, would be key to understanding the connection between and within pathways. The implications to cancer therapeutics and/or prevention would be significant.